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In this study, the effect of novel taxane SB-T-1216 and paclitaxel were compared on drug-sensitive MDA-MB-435 and drug-resistant NCI/ADR-RES human breast cancer cells.
Cell growth and survival were evaluated after 96-hour incubation with tested concentrations of taxanes. The effect on the formation of microtubule bundles was assessed employing fluorescence microscopy and on the cell cycle employing flow cytometric analysis. The activity of caspases was assessed employing commercial colorimetric kits.
The IC50 (concentration resulting in 50% of living cells in comparison with the control) of SB-T-1216 in drug-sensitive cells was 0.6 nM versus 1 nM for paclitaxel. However, the IC50 of SB-T-1216 in drug-resistant cells was 1.8 nM versus 300 nM for paclitaxel. Both SB-T-1216 and paclitaxel at death-inducing concentrations induced the formation of microtubule bundles in drug-sensitive as well as drug-resistant cells. Cell death induced in drug-sensitive and drug-resistant cells by paclitaxel was associated with the accumulation of cells in the G2/M phase. On the contrary, cell death induced by SB-T-1216 took place without the accumulation of cells in the G2/M phase but with a decreased number of G1 cells and the accumulation of hypodiploid cells. Both SB-T-1216 and paclitaxel activated caspase-3, caspase-9, caspase-2 and caspase-8 in drug-sensitive as well as drug-resistant cells.
Cell death induced by both paclitaxel and novel taxane SB-T-1216 in breast cancer cells is associated with caspase activation and with the formation of interphase microtubule bundles. Novel taxane SB-T-1216, but not paclitaxel, seems to be capable of inducing cell death without the accumulation of cells in the G2/M phase.
Classical taxanes, such as paclitaxel (Taxol®) and docetaxel (Taxotere®), have been successfully used in the therapy not only of breast and ovarian cancer, but also of other types of cancer. Paclitaxel was originally isolated from the bark of the Pacific Yew (Taxus brevifolia) white docetaxel is a semisynthetic taxane (1–5).
Microtubules are polymers of tubulin heterodimers containing α and β subunits (6). Microtubules comprise one of the major components of the cytoskeleton of cells and are involved in many cellular processes including mitotic division (7). At the onset of mitosis, cytoplasmic microtubules undergo dramatic rearrangement to form mitotic spindles. Regular bipolar spindles play a pivotal role in the precise segregation of chromosomes. On the other hand, aberrant multipolar spindles are not compatible with cell viability. Most cells derived from defective mitosis are supposed to undergo apoptosis (8). Taxanes are known as mitotic poisons due to their binding to the β subunit of the tubulin heterodimer. This binding accelerates the polymerization of tubulin, stabilizes microtubules and inhibits their depolymerization (6, 9–11). Thus the interaction of taxanes with microtubules results in the formation of microtubule bundles in interphase cells and the formation of asters instead of mitotic spindles during mitosis. In this way, taxanes are thought to block progression through the M-phase of the cell cycle (12, 13). However, the relationship of the mitotic arrest to the induction of cell death by taxanes is unclear (14–16).
Novel synthetic paclitaxel analogs represent new generation taxanes. Some of them have been found to be more effective or more suitable than paclitaxel and docetaxel in the treatment of various types of cancer cells. They are particularly more effective in drug-resistant cancer cells (4, 17–21). Thus novel taxanes provide us with a better chance in the therapy of such types of cancer as breast and ovarian. Some novel taxanes have been found to exhibit a significant activity towards microtubules (13, 19).
We have shown previously that novel taxane SB-T-1216 is found to be more effective, particularly against drug-resistant breast cancer cells, than paclitaxel (18). In the present study, the effect of novel taxane SB-T-1216 and paclitaxel on the formation of microtubule bundles and cell cycle progression, as well as on the activity of caspase-3, caspase-9, caspase-2 and caspase-8, were compared in drug-sensitive MDA-MB-435 and drug-resistant NCI/ADR-RES human breast cancer cells.
Paclitaxel was obtained from Bristol-Myers Squibb (Princeton, NJ, USA). SB-T-1216 was synthesized in the laboratory of Professor I. Ojima (Stony Brook, NY, USA) (20). Indocarbocyanate (Cy3)-conjugated monoclonal anti-tubulin antibody (recognizing an epitope in the C-terminal of β-tubulin) was obtained from Sigma (St. Louis, MO, USA).
The human breast carcinoma cell lines MDA-MB-435 and NCI/ADR-RES, were obtained from the National Cancer Institute (Frederick, MD, USA). Cells were maintained in a culture medium at 37 °C in a humidified atmosphere of 5% CO2 in air. The culture medium was RPMI-1640 medium containing extra L-glutamine (300 μg/ml), sodium pyruvate (110 μg/ml), HEPES (15 mM), penicillin (100 U/ml), and streptomycin (100 μg/ml) as described elsewhere (22) supplemented with 10% fetal bovine serum (M. Kysilka, Brno, Czech Republic). For the experiments, cells were harvested employing trypsin (0.2%) + EDTA (0.02%) in PBS.
Cells maintained in the culture medium were harvested by low-speed centrifugation, washed with the culture medium and then seeded at 1×104 cells/100 μl of medium into the wells of a 96-well plastic plate. Cell growth and survival were evaluated after 96 h of incubation in the culture medium without taxane (control) and at a range of concentrations (0.01–100 nM) of SB-T-1216. The number of living cells was determined by hemacytometer counting after staining with trypan blue (23).
Cells grown in the culture medium were harvested by low-speed centrifugation, washed with the culture medium and seeded at 3×106 cells/15 ml of culture medium into plastic culture dishes for 24-h preincubation, allowing cells to attach. The medium was changed using culture medium cells without taxane (control) and at selected concentrations of paclitaxel (30 nM for MDA-MB-435 cells, 3000 nM for NCI/ADR-RES cells) or SB-T-1216 (10 nM for MDA-MB-435 cells, 100 nM for NCI/ADR-RES). After the required incubation period (24, 48, 72 h), the cells were harvested by low-speed centrifugation, stained and analyzed as described elsewhere (23).
For immunofluorescence microscopy, cells were seeded in culture medium onto glass slides for 24-h preincubation, allowing cells to attach. The medium was replaced after this time with culture medium without taxane (control) or at selected concentrations of paclitaxel or SB-T-1216. After incubation (24 h) attached cells were rinsed twice with microtubule-stabilizing buffer (MSB, 20 mM 2-(N-morpholino)ethanesulfonic acid adjusted to pH 6.9 with KOH, 2 mM EGTA, 2 mM MgCl2, and supplemented with 4% PEG 6000). Cells were fixed for 20 min at 37°C with 3% formaldehyde in MSB and then extracted for 4 min with 0.5% Triton®X-100 in MSB. After rinsing the glass slides with MSB, cells were incubated with the Cy3-conjugated anti-tubulin antibody (1:500) for 45 min at room temperature. The preparations were mounted in Mowiol 4–88 supplemented with 1 μg/ml 4,6-diamidino-2-phenylindole (DAPI) used to label cell nuclei. Visualization was carried out with an Fluorescence A70 Provis microscope (Olympus, Hamburg, Germany) and images were captured and processed using a cooled CCD camera SensiCam (Olympus).
Harvested cells were seeded at 5×106 cells/25 ml of culture medium into plastic culture flasks for 24-h preincubation allowing cells to attach after which the medium was changed to a relevant test medium. After 24 h of incubation in culture medium without taxane (control) or with paclitaxel or SB-T-1216, the cells were harvested by low-speed centrifugation and analyzed as described elsewhere (16, 24). Commercial colorimetric kits, Caspase-3/CPP32 Colorimetric Protease Assay, Caspase-9/Mch6/Apaf-3 Colorimetric Protease Assay and Caspase-8/FLICE Colorimetric Protease Assay from Biosource (Camarillo, CA, USA), as well as solutions from a Caspase-3 Colorimetric Protease Assay kit (Biosource) combined with the chromogenic caspase-2 substrate from Alexis Biochemicals (Lausen, Switzerland) were used. The total protein content in samples was determined by the bicinchoninic acid assay (25).
The statistical significance of differences was determined using Student’s t-test. P<0.05 was considered statistically significant.
The effect of paclitaxel on drug-sensitive MDA-MB-435 cells and drug-resistant NCI/ADR-RES cells was tested previously (16). Paclitaxel at a concentration of 3 nM and higher induced death of MDA-MB-435 cells within 96 h of incubation, i.e. the number of living cells after the incubation period was lower than the number of cells of the inoculus. The IC50 (concentration of taxane resulting in 50% of living cells in comparison with the control after 96 h of incubation) of paclitaxel was 1 nM. On the contrary, paclitaxel induced the death of NCI/ADR-RES cells at a concentration of 1000 nM and higher. The IC50 of paclitaxel was 300 nM. Approximately 300-fold higher concentration of paclitaxel was required to induce the death of drug-resistant NCI/ADR-RES cells than of drug-sensitive MDA-MB-435 cells. On the basis of these data, 30 nM paclitaxel for drug-sensitive MDA-MB-435 cells and 3,000 nM paclitaxel for drug-resistant NCI/ADR-RES cells were employed as death-inducing concentrations, i.e. the lowest concentrations with full death-inducing effect (16).
The effect of SB-T-1216 on the growth and survival of drug-sensitive MDA-MB-435 and drug-resistant NCI/ADR-RES cells was tested at a wide range of concentrations (0.01–100 nM). SB-T-1216 induced the death of MDA-MB-435 cells within 96 h of incubation at a concentration of 3 nM and higher. The IC50 of SB-T-1216 for MDA-MB-435 cells was 0.6 nM. In the case of NCI/ADR-RES cells, SB-T-1216 induced cell death at a concentration of 10 nM and higher. The IC50 of SB-T-1216 for NCI/ADR-RES cells was 1.8 nM (Figure 1). The data showed that only about 3-fold higher concentrations of SB-T-1216 were required to induce death in drug-resistant NCI/ADR-RES cells than in drug-sensitive MDA-MB-435 cells. The result indicates that SB-T-1216 was much more effective in drug-resistant NCI/ADR-RES cells than paclitaxel. On the basis of these data, the death-inducing concentrations of SB-T-1216, i.e. 10 nM for drug-sensitive MDA-MB-435 cells and 100 nM for drug-resistant NCI/ADR-RES cells were used. The death-inducing concentrations of paclitaxel and SB-T-1216 were used in further studies with MDA-MB-435 and NCI/ADR-RES cells.
In drug-sensitive MDA-MB-435 cells, paclitaxel at 30 nM as well as SB-T-1216 at 10 nM induced the formation of microtubule bundles within 24 h of incubation (Figure 2). Similarly in drug-resistant NCI/ADR-RES cells, both paclitaxel and SB-T-1216 at 3,000 nM and 100 nM also induced the formation of microtubule bundles within 24 h of incubation (Figure 2).
Flow cytometric analysis, after propidium iodide staining, showed that the application of paclitaxel at 30 nM resulted in nearly total accumulation of drug-sensitive MDA-MB-435 cells in the G2/M phase of the cell cycle after 24 h of incubation. On the contrary, the application of SB-T-1216 at 10 nM was without any accumulation of the cells in the G2/M phase. The G1 peak was significantly decreased and the accumulation of near-G1 hypodiploid cells/particles was apparent (Figure 3).
Similarly, the application of paclitaxel at 3,000 nM resulted in the accumulation of drug-resistant NCI/ADR-RES cells in the G2/M phase after 24 h of incubation. The application of SB-T-1216 at 100 nM was again without accumulation of the cells in the G2/M phase. The G1 peak disappeared and the accumulation of near-G1 hypodiploid cells/particles was apparent (Figure 3). This characteristic effect of 100 nM SB-T-1216 without the accumulation in the G2/M phase was found after 24, 48 and 72 hour of incubation. The G2 peak even disappeared within 72-hour incubation (Figure 4).
The employed colorimetric assays showed that after 24 h of incubation with paclitaxel or SB-T-1216 at the death-inducing concentrations (30 nM or 10 nM, respectively), the activity of caspase-3 increased significantly in drug-sensitive MDA-MB-435 cells. Caspase-3 activity increased approximately 7.5-fold after the application of paclitaxel (P<0.01) and approximately 5.5-fold after the application of SB-T-1216 (P<0.05). Similarly, caspase-3 activity increased significantly (P<0.01) in drug-resistant NCI/ADR-RES cells after incubation with paclitaxel or SB-T-1216 at the death-inducing concentrations (3,000 nM or 100 nM). The activity of caspase-3 increased approximately 4.5-fold in the case of paclitaxel and approximately 2-fold in the case of SB-T-1216 (Figure 5A).
The activity of caspase-9 also increased significantly in both drug-sensitive MDA-MB-435 (P<0.05) and drug-resistant NCI/ADR-RES (P<0.01) cells after incubation with the death-inducing concentration of paclitaxel or SB-T-1216. The increase was approximately 2.5- to 4-fold for paclitaxel and approximately 3- to 5-fold for SB-T-1216 (Figure 5B).
Concerning caspase-2 activity in drug-sensitive MDA-MB-435 cells, a 7.5-fold increase was detected after incubation with the death-inducing concentration of paclitaxel (P<0.05) and a 4.5-fold increase after incubation with SB-T-1216 (P<0.05). In drug-resistant NCI/ADR-RES cells, we detected a smaller but still statistically significant 2-fold increase of caspase-2 activity after incubation with paclitaxel (P<0.01) and approximately 4.5-fold increase after incubation with SB-T-1216 (P<0.01) (Figure 5C).
A significant (P<0.01) approximately 4.5-fold increase of caspase-8 activity was detected in drug-sensitive MDA-MB-435 cells after incubation with death-inducing concentrations of both paclitaxel and SB-T-1216. In drug-resistant NCI/ADR-RES cells, again a smaller but still statistically significant 1.5-fold increase of caspase-8 activity was detected for paclitaxel (P<0.05) and an approximately 3-fold increase for SB-T-1216 (P<0.01) (Figure 5D).
Taxanes are known to modify microtubule dynamics leading to the formation of interphase microtubule bundles and aberrant mitotic spindles. These events are believed to result in blocking of progression through the M-phase of the cell cycle (6, 9, 10, 13). It is also supposed that mitotic arrest could represent a mechanism by which taxanes induce cell death. However, this question has not been clearly answered yet (14–16).
We reported previously that novel taxane SB-T-1216 was more effective than classical taxanes against breast cancer cells (18). Our previous data (16) together with data of the present study (see Figure 1) demonstrate that SB-T-1216 is much more effective than paclitaxel against drug-resistant breast cancer cells and that SB-T-1216 could represent a potentially powerful tool for the treatment of drug-resistant breast cancer. Therefore, we were curious about the mechanism of the higher efficiency of SB-T-1216 and thus we compared the effects of SB-T-1216 and paclitaxel on the formation of interphase microtubule bundles, cell cycle progression and also on the activity of caspase-3, caspase-9, caspase-2 and caspase-8 in both drug-sensitive MDA-MB-435 and drug-resistant NCI/ADR-RES human breast cancer cells.
Both paclitaxel and SB-T-1216 at death-inducing concentrations induced the formation of interphase microtubule bundles in drug-sensitive MDA-MB-435 cells during 24-h incubation. Similarly paclitaxel and SB-T-1216 induced the formation of interphase microtubule bundles in drug-resistant NCI/ADR-RES cells (see Figure 2). It was shown previously that some novel taxanes can exhibit a signitificant activity towards microtubules (11, 13, 19, 21) and that they could interact with microtubules differently than paclitaxel (13). However, it does not seem that SB-T-1216 at death-inducing concentrations exerts different effect on the formation of microtubule bundles than paclitaxel in tested drug-sensitive and drug-resistant breast cancer cells. Thus our findings suggest that the induction of cell death by both taxanes in breast cancer cells is associated with analogous formation of interphase microtubule bundles.
Cell death induced in both MDA-MB-435 cells and NCI/ADR-RES cells at the death-inducing concentration of SB-T-1216 resulted in a markedly different cell cycle distribution from that of paclitaxel. The effect of paclitaxel was characterized by the accumulation of cells in the G2/M phase. On the contrary, the effect of SB-T-1216 was not associated with the accumulation in the G2/M phase and was characterized by the accumulation of hypodiploid cells (see Figure 3 and and4).4). These findings suggest that novel taxane SB-T-1216 at death-inducing concentrations, but not paclitaxel, induces cell death via a pathway different from that involving M-phase block. Some previous studies have suggested that taxanes can induce apoptosis independent of mitotic arrest (14, 26–28). This is usually related to lower taxane concentrations, while higher taxane concentrations cause cell death connected with mitotic arrest (27–29). Thus, it seems that mitotic arrest does not represent an indispensable prerequisite for taxane-induced cell death. Such indication is also supported by the finding that baccatin III, which contains the core taxane ring, can induce apoptosis independent of G2/M arrest (14). Our results suggest that the ability of SB-T-1216, but not of paclitaxel, to switch on the pathway independent of mitotic arrest in drug-resistant cells can explain, at least partly, the significantly higher potency of SB-T-1216 against drug-resistant cells.
The key executioner caspase-3 and upstream caspase-9 were activated in both drug-sensitive MDA-MB-435 and drug-resistant NCI/ADR-RES cells when cell death was induced by paclitaxel (16) as well as by SB-T-1216 (see Figure 5A and 5B). This fact indicates that the cell death induced by both taxanes could be related to the mitochondrial pathway of caspase activation as it was also suggested by others (30–34). However, significant activation of caspase-2 and caspase-8 was also found in both cell lines after the application of both taxanes at the death-inducing concentration (see Figure 5C and 5D). The role of caspase-2 in apoptosis induction is not clear yet but several lines of evidence point at caspase-2 as being a major player in apoptosis induction (35, 36). Several studies demonstrated caspase-2 activation in various types of cancer cells after apoptosis induction by taxanes (37–39). The involvement of caspase-2 activation in apoptosis of breast cancer cells induced by various stimuli was also demontrated (40–42). Concerning caspase-8 activation, the activation was shown during taxane-induced apoptosis in lymphoma and melanoma cells (38, 39) and during apoptosis induction by HOXA5 in breast cancer cells (40). The activation of caspase-8 suggests that the death receptor pathway of caspase activation could also be somehow involved in cell death induced by taxanes. Considering the activation of all discussed caspases, functional sequence of the activation of individual caspases remains to be elucidated in order to understand better mechanisms involved in apoptosis induction by taxanes in breast cancer cells.
Taken together, we conclude that cell death induced by both of the tested taxanes, namely paclitaxel and novel taxane SB-T-1216, in breast cancer cells is associated with the activation of several caspases and with the formation of interphase microtubule bundles. However, we suggest that novel taxane SB-T-1216, but not paclitaxel, can induce cell death that is not directly related to the accumulation of cells in the G2/M phase, via a pathway independent of M-phase block. The ability of SB-T-1216 to switch on such a pathway in drug-resistant cells could help to explain its significantly higher potency against those cells.
This work was supported by grant NR9426-3/2007 from the IGA, Ministry of Health of the Czech Republic, by grant 301/09/0362 from the Grant Agency of the Czech Republic, by grant 204/05/H023 from the Grant Agency of the Czech Republic, a grant from the National Cancer Institute, USA (CA103314 to I.O.) and a Faculty Development Award from the New York State Office of Science, Technology & Academic Research (to I.O.).